Methods for evaluating renal toxicity

Abstract

The present disclosure provides a method for evaluating the renal toxicity of a test substance as an improved in vitro evaluation system for evaluating the renal toxicity of a substance, such as a compound, the method comprising the step of contacting the test substance with a renal organoid, higher cytotoxicity of the test substance indicates that the test substance may have renal toxicity.

Description

Technical Field

[0001] The present disclosure relates to a method for evaluating the nephrotoxicity of a test substance. More particularly, the present disclosure relates to an in vitro nephrotoxicity evaluation method using kidney organoids. Background of the Invention

[0003] An in vitro evaluation system using a renal proximal tubule epithelial cell line (RPTEC) has been proposed for evaluating the nephrotoxicity of substances such as compounds (see, for example, Non-Patent Document 1). In the in vitro evaluation system using RPTEC, it is difficult to evaluate the toxicity to renal constituent cells other than renal proximal tubules (such as glomerular epithelial cells), and thus, in some cases, results different from those of the toxicity reaction in a living kidney including other cells than renal proximal tubules are obtained.

[0004] Related to the present disclosure, Non-Patent Documents 2 to 4 disclose techniques for inducing kidney organoids (KiO) from stem cells.

[0005] Non-Patent Document

[0006] Non-Patent Document 1: "Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules", Marianne K. Vormann, et al., The AAPS Journal, 2018, 20, Art. No. 90

[0007] Non-Patent Document 2: "Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis", Minoru Takasato et al., Nature, 2015, Vol. 526, pp. 564 - 568

[0008] Non-Patent Document 3: "Generation of kidney organoids from human pluripotent stem cells", Minoru Takasato et al., Nature Protocols, 2016, Vol. 11, pp. 1681 - 1692

[0009] Non-Patent Document 4: "Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk", Sara E. Howden et al., Cell Stem Cell, 2021, Vol. 28, No. 4, pp. 671-684 Summary of the Invention

[0010] Technical Problem

[0011] The main object of the present disclosure is to provide an improved in vitro evaluation system for evaluating the nephrotoxicity of substances such as compounds.

[0012] Solution to the Problem

[0013] To solve this problem, the present disclosure provides the following [1] to

[32] :

[0014] [1] A method for evaluating the nephrotoxicity of a test substance, the method comprising the step of contacting the test substance with a kidney organoid, wherein a higher cytotoxicity of the test substance compared to a control substance indicates that the test substance may have nephrotoxicity.

[0015] [2] A method for screening a test substance that may have nephrotoxicity, the method comprising the following steps: (1) contacting the test substance with a kidney organoid; and (2) selecting a test substance that shows higher cytotoxicity compared to a control substance.

[0016] [3] A method for screening a test substance that may have no nephrotoxicity or may have low nephrotoxicity, the method comprising the following steps: (1) contacting the test substance with a kidney organoid; and (2) selecting a test substance that shows no cytotoxicity or lower cytotoxicity compared to a control substance.

[0017] [4] The method according to any one of [1] to [3], wherein the kidney organoid is derived from human induced pluripotent stem cells.

[0018] [5] The method according to any one of [1] to [4], wherein the kidney organoid is rich in glomerular epithelial cell-like cells.

[0019] [6] The method according to [5], wherein 30% to 80% of the cells contained in the kidney organoid are glomerular epithelial cell-like cells.

[0020] [7] The method according to [5] or [6], further comprising step (a) of culturing pluripotent stem cells in the presence of a 9.0 to 10.5 μM GSK3β inhibitor to obtain renal organoids enriched in glomerular epithelial cell-like cells.

[0021] [7a] The method according to [7], wherein the method comprises step (a) before step (1).

[0022] [8] The method according to [7] or [7a], wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile).

[0023] [9] The method according to any one of [1] to [4], wherein the renal organoids are enriched in renal proximal tubule epithelial cell-like cells.

[0024]

[10] The method according to [9], wherein 5% to 30% of the cells contained in the renal organoids are renal proximal tubule epithelial cell-like cells.

[0025]

[11] The method according to [9] or

[10] , further comprising step (b) of culturing pluripotent stem cells in the presence of a 5.5 to 8.5 μM GSK3β inhibitor to obtain renal organoids enriched in renal proximal tubule epithelial cell-like cells.

[0026]

[12] The method according to

[11] , wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile).

[0027]

[13] A method for predicting the site in the kidney where a test substance shows toxicity, the method comprising the following steps: (A) contacting the test substance with renal organoids enriched in glomerular epithelial cell-like cells; (B) contacting the test substance with renal organoids enriched in renal proximal tubule epithelial cell-like cells; and (C) comparing the cytotoxicity obtained in step (A) with the cytotoxicity obtained in step (B) to predict that the site showing toxicity is the glomerulus when the cytotoxicity obtained in step (A) is higher, and predicting that the site showing toxicity is the renal proximal tubule when the cytotoxicity obtained in step (B) is higher.

[0028]

[14] The method according to

[13] , wherein 30% to 80% of the cells contained in the kidney organoids rich in glomerular epithelial cell-like cells are glomerular epithelial cell-like cells, and 5% to 30% of the cells contained in the kidney organoids rich in renal proximal tubule epithelial cell-like cells are renal proximal tubule epithelial cell-like cells.

[0029]

[15] The method according to

[13] or

[14] , the method further comprising the steps of: (a) culturing pluripotent stem cells in the presence of a 9.0 to 10.5 μM GSK3β inhibitor to obtain kidney organoids rich in glomerular epithelial cell-like cells; and (b) culturing pluripotent stem cells in the presence of a 5.5 to 8.5 μM GSK3β inhibitor to obtain kidney organoids rich in renal proximal tubule epithelial cell-like cells.

[0030]

[16] The method according to

[15] , wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile).

[0031]

[17] A method for screening the nephrotoxicity of a drug candidate compound, the method comprising the steps of: (1) contacting a test substance with kidney organoids; and (2) selecting a test substance that shows no cytotoxicity or lower cytotoxicity compared to a control substance as a drug candidate compound.

[0032]

[18] The method according to

[17] , wherein the kidney organoids are derived from human induced pluripotent stem cells.

[0033]

[19] The method according to

[17] or

[18] , wherein the kidney organoids are rich in glomerular epithelial cell-like cells.

[0034]

[20] The method according to

[19] , wherein 30% to 80% of the cells contained in the kidney organoids are glomerular epithelial cell-like cells.

[0035]

[21] The method according to

[19] or

[20] , the method further comprising step (a) of culturing pluripotent stem cells in the presence of a 9.0 to 10.5 μM GSK3β inhibitor to obtain kidney organoids rich in glomerular epithelial cell-like cells.

[0036]

[22] The method according to

[21] , wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile).

[0037]

[23] The method according to

[17] or

[18] , wherein the renal organoid is rich in renal proximal tubule epithelial cell-like cells.

[0038]

[24] The method according to

[23] , wherein 5% to 30% of the cells contained in the renal organoid are renal proximal tubule epithelial cell-like cells.

[0039]

[25] The method according to

[23] or

[24] , the method further comprising the step (b) of culturing pluripotent stem cells in the presence of a 5.5 to 8.5 μM GSK3β inhibitor to obtain a renal organoid rich in renal proximal tubule epithelial cell-like cells.

[0040]

[26] The method according to

[25] , wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile).

[0041]

[27] A kit for evaluating the nephrotoxicity of a test substance, the kit comprising a renal organoid.

[0042]

[28] The kit according to

[27] , wherein the renal organoid is rich in glomerular epithelial cell-like cells.

[0043]

[29] The kit according to

[27] , wherein the renal organoid is rich in renal proximal tubule epithelial cell-like cells.

[0044]

[30] A method for generating a renal organoid rich in glomerular epithelial cell-like cells, the method comprising the step (a) of culturing pluripotent stem cells in the presence of a 9.0 to 10.5 μM GSK3β inhibitor.

[0045]

[31] A method for generating a renal organoid rich in renal proximal tubule epithelial cell-like cells, the method comprising the step (b) of culturing pluripotent stem cells in the presence of a 5.5 to 8.5 μM GSK3β inhibitor.

[0046]

[32] The production method according to

[30] or

[31] , wherein the GSK3β inhibitor is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethyl]amino]nicotinonitrile).

[0047] [Definition]

[0048] The term "renal organoid" refers to a three-dimensional structure that constitutes the renal tissue of a living body and contains at least one or more cell populations.

[0049] The term "glomerular epithelial cell-like cell" refers to a cell induced from a stem cell (such as a pluripotent stem cell) and having the same properties as a glomerular epithelial cell. The phrase "the same properties as a glomerular epithelial cell" means being positive for specific staining against glomerular epithelial cells and means expressing glomerular epithelial cell marker genes therein. Glomerular epithelial cells are positive for CCND1, CDH6, EMX2, and SOX4.

[0050] The term "renal proximal tubule epithelial cell-like cell" refers to a cell induced from a stem cell (such as a pluripotent stem cell) and having the same properties as a renal proximal tubule epithelial cell. The phrase "the same properties as a renal proximal tubule epithelial cell" means being positive for specific staining against renal proximal tubule epithelial cells and means expressing renal proximal tubule epithelial cell marker genes therein. Renal proximal tubule epithelial cells are positive for LTL, DAB2, CUBN, and SLC34A1.

[0051] The term "intermediate mesoderm" refers to an embryo generated from the mesoderm during ontogeny and being cells capable of differentiating into the pronephros, mesonephros, mesonephric duct, metanephros, adrenal cortex, and gonad. A type of embryo generated from the mesoderm during ontogeny and being cells capable of differentiating into the pronephros, mesonephros, mesonephric duct, metanephros, adrenal cortex, and gonad. Intermediate mesoderm is positive for OSR1 (odd-skipped related 1).

[0052] The term "GSK3β inhibitor" refers to a substance having inhibitory activity against GSK3β (glycogen synthase kinase 3β). GSK3 (glycogen synthase kinase 3) is a class of serine / threonine protein kinases and is involved in a large number of signal transduction pathways related to glycogenogenesis, apoptosis, and stem cell maintenance. GSK3 has two subtypes: GSK3α and GSK3β. There is no particular limitation on the GSK3β inhibitor as long as it has GSK3β inhibitory activity, and it can be a substance having GSK3α inhibitory activity in addition to GSK3β inhibitory activity.

[0053] The term "culture" refers to maintaining, proliferating (growing), and / or differentiating cells in an in vitro environment. The term "culture" refers to maintaining, proliferating (growing), and / or differentiating cells outside of a tissue or the body, such as in a cell culture dish or flask.

[0054] The term "pluripotency" refers to the ability to differentiate into tissues and cells with various different forms and functions and into cells of any system of the three germ layers. "Pluripotency" does not include the ability to differentiate into the blastoderm and thus does not have the ability to develop an individual, and thus is distinguished from "totipotency" which is the ability to differentiate into all tissues of a living body including the blastoderm.

[0055] The term "multipotency" refers to the ability of cells to differentiate into a limited number of multiple lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0056] The term "marker" refers to a "marker protein" or a "marker gene", and means a protein or its gene that is specifically expressed on the cell surface, in the cytoplasm, and / or in the nucleus of cells of a specified cell type. A marker can be a positive selection marker or a negative selection marker. Preferably, the marker is a cell surface marker, and especially when a cell surface positive selection marker is used, viable cells can be concentrated, separated, and / or detected.

[0057] A marker protein can be detected by immunoassays (such as ELISA, immunostaining, and flow cytometry) using an antibody specific for the marker protein. As an antibody specific for the marker protein, an antibody that binds to a specific amino acid sequence of the marker protein, or an antibody that binds to a specific sugar chain or the like bound by the marker protein can be used. When the marker protein is expressed in cells but not on the cell surface (for example, a transcription factor or its subunit), the target marker protein can be detected by co-expressing a reporter protein with the marker protein and detecting the reporter protein (for example, Non-Patent Document 4). This method is preferably used when no suitable cell surface marker is found. Detection of a marker gene can be carried out by nucleic acid amplification methods and / or nucleic acid detection methods known in the art, such as RT-PCR, microarray, biochip, and RNAseq methods.

[0058] The term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by an intracellular promoter.

[0059] The term "being positive" or "expressing" means that a protein or a gene is expressed at a level detectable by methods known in the art. Detection of a protein can be carried out by immunoassays (such as ELISA, immunostaining, or flow cytometry) using an antibody. When a protein is expressed in cells but not on the cell surface (for example, a transcription factor or its subunit), the target protein can be detected by co-expressing a reporter protein with the target protein and detecting the reporter protein. Detection of a gene can be carried out by nucleic acid amplification methods and / or nucleic acid detection methods, such as RT-PCR, microarray, biochip, and RNAseq methods.

[0060] The term "being negative" or "not expressing" means that the expression level of a protein or a gene is below the detection limit of all or any of the above-known methods. The detection limit of the expression of a protein or a gene can vary between methods.

[0061] The term "comprise(s)" or "comprising" means including but not limited to the elements that follow the term. Thus, it is recommended to include the elements that follow the term, but it is not recommended to exclude another optional element.

[0062] Advantageous effects of the present invention

[0063] The present disclosure provides an improved in vitro evaluation system for evaluating the nephrotoxicity of substances such as compounds. Brief description of the drawings

[0064] Figure 1 Figure 1 Illustrate the experimental protocols for steps 2-3 and 3 in the overview of the induction of differentiation from iPSC to KiO.

[0065] Figure 2 Figure 2 Show the CHIR addition period conditions (1) to (3) in Example 1.

[0066] Figure 3 Figure 3 Show the fluorescence microscopy images of KiO induced under the condition of changing the CHIR addition period (Example 1).

[0067] Figure 4 Figure 4 Show the fluorescence microscopy images of KiO induced under the condition of changing the CHIR addition concentration (Example 1).

[0068] Figure 5 Figure 5 Show the phase-contrast microscopy images of cells with a renal tubule-like shape induced in KiO (Example 1).

[0069] Figure 6 Figure 6 Show the gene expression patterns of KiO induced at CHIR addition concentrations of 6 μM, 8 μM, and 10 μM.

[0070] Figure 7 Figure 7 Show the evaluation results of the cytotoxicity of cyclosporine A obtained by using KiO prepared by the method described in Example 2 and the renal proximal tubule epithelial cell line (RPTEC) (Example 3).

[0071] Figure 8 Figure 8 ​​​​​​​​​​​​​​​​Show the evaluation results of the cytotoxicity of cyclosporin A obtained based on the intracellular ATP amount, using KiO (tubule-rich) with a high ratio of renal proximal tubule epithelial cell-like cells, KiO (glomerulus-rich) with a high ratio of glomerular epithelial cell-like cells, and KiO (intermediate) with an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells generated by the method described in Example 1 (Example 3).

[0072] Figure 9 Figure 9 Show the evaluation results of the cytotoxicity of cyclosporin A obtained based on the LDH leakage amount, using KiO (tubule-rich) with a high ratio of renal proximal tubule epithelial cell-like cells, KiO (glomerulus-rich) with a high ratio of glomerular epithelial cell-like cells, and KiO (intermediate) with an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells generated by the method described in Example 1 (Example 3).

[0073] Figure 10 Figure 10 Show the evaluation results of the cytotoxicity of cyclosporin A obtained based on the amount of secreted KIM-1, using KiO (tubule-rich) with a high ratio of renal proximal tubule epithelial cell-like cells, KiO (glomerulus-rich) with a high ratio of glomerular epithelial cell-like cells, and KiO (intermediate) with an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells generated by the method described in Example 1 (Example 3). Detailed Description

[0074] Now, preferred embodiments for implementing the present disclosure will be described. Note that the embodiments described below are merely examples of representative embodiments of the present disclosure, and the scope of the present disclosure should not be construed narrowly.

[0075] 1. Method for Evaluating the Nephrotoxicity of a Test Substance

[0076] The method for evaluating the nephrotoxicity of a test substance of the present disclosure includes the step of contacting the test substance with a renal organoid, and when the test substance shows higher cytotoxicity compared to a control substance, it is determined that the test substance may have nephrotoxicity.

[0077] [Test Substance]

[0078] ​​​​The test substance is not particularly limited and can be a compound, such as an organic low-molecular-weight compound; a metal complex; a polymer (including an aptamer), such as a peptide, a protein, an antibody, or a nucleic acid; a sugar; a lipid; a tissue extract; a cell extract; a cell culture supernatant; a plant extract; a microbial product; or a biological material, such as blood or urine, and particularly, can be a drug candidate compound. The test substance can be a synthetic compound or a compound extracted from a natural product.

[0079] [Pluripotent stem cell]

[0080] Renal organoids can be induced from stem cells in vitro. The induction of renal organoids from pluripotent stem cells can be carried out according to conventionally known methods (for example, those described in Non-Patent Documents 2 to 4).

[0081] The term "pluripotent stem cell" refers to a stem cell that is capable of differentiating into tissues or cells having various different biological forms and functions and has the ability to differentiate into cells of any system of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells include embryonic stem cells (ESCs), embryonic stem cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells, embryonic germ cells, and induced pluripotent stem cells (also referred to as "iPSCs" in this article).

[0082] The term "multipotent stem cell" refers to a stem cell that has the ability to differentiate into cells of a limited number of multiple systems. Examples of multipotent stem cells include dental pulp stem cells, stem cells derived from oral mucosa, hair follicle stem cells, and adult stem cells derived from cultured fibroblasts or bone marrow stem cells.

[0083] The pluripotent stem cell is preferably an ESC or an iPSC.

[0084] As "ESC", especially as mouse ESC, various mouse ESC lines established by inGenious Targeting Laboratory Inc., RIKEN (Institute of Physical and Chemical Research), etc. can be used, and as human ESC, various human ESC lines established by Wisconsin University, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used. For example, as human ESC lines, CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 distributed by ESI Bio, H1 and H9 distributed by WiCell Research Institute Inc., and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 distributed by RIKEN can be used.

[0085] The term "iPSC" refers to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells with specific factors (nuclear reprogramming factors) introduced therein. Currently, there are various iPSCs, and not only the iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K., Yamanaka S., Cell (2006) 126:663-676) can be used, but also human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K., Yamanaka S. et al., Cell (2007) 131:861-872), Nanog-iPSCs established using the expression of Nanog as an indicator after introducing the four factors (Okita, K., Ichisaka, T. and Yamanaka S. (2007), Nature 448, 313-317), iPSCs generated by a method without c-Myc (Nakagawa M., Yamanaka S. et al., Nature Biotechnology (2008) 26, 101-106), and iPSCs established by introducing six factors by a virus-free method (Okita K. et al., Nat. Methods May 2011; 8(5):409-12; Okita K. et al., Stem Cells 31(3):458-66). In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, generated by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920), induced pluripotent stem cells generated by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), induced pluripotent stem cells generated by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc. can be used.

[0086] In addition, any of the induced pluripotent stem cells known in the art described in all published papers (such as Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol. 3, No. 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7, 795-797) or patents (such as Japanese Patent Laid-Open No. 2008-307007, Japanese Patent Laid-Open No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0087] As the induced pluripotent stem cell line, various iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPSC lines include HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A and Nips-B2 of RIKEN, and 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1 and 1231A3 of Kyoto University, etc., among which 1231A1 and 1231A3 are preferred, and 1231A3 is more preferred.

[0088] [Inducing kidney organoids from pluripotent stem cells]

[0089] Inducing kidney organoids from pluripotent stem cells can be carried out as follows, especially when using human iPSCs.

[0090] First, human iPSCs are cultured in a medium containing a GSK3β inhibitor, then cultured in a medium containing FGF9 and heparin, the obtained product is dissociated into single cells, and about 1×10 4 to 1×10 5 cells are further cultured in a medium containing FGF9 and heparin to form cell aggregates (intermediate mesoderm spheroids) (step i).

[0091] Subsequently, the medium containing the GSK3β inhibitor is sequentially replaced with a medium containing FGF9 and heparin, and the intermediate mesoderm spheroids are cultured at the air-liquid interface to obtain kidney organoids (step ii).

[0092] The concentration of the GSK3β inhibitor in step i is, for example, 0.1 to 30 μM, preferably 1 to 20 μM, more preferably 6 to 10 μM, and particularly preferably 8 μM.

[0093] The concentration of FGF9 is, for example, 0.1 ng / ml to 1 μg / ml, preferably 1 to 500 ng / ml, more preferably 10 to 300 ng / ml, and particularly preferably about 200 ng / ml.

[0094] The concentration of heparin is, for example, 0.01 to 100 μg / ml, preferably 0.1 to 10 μg / ml, and particularly preferably about 1 μg / ml.

[0095] The culturing period in the medium containing the GSK3β inhibitor is, for example, 3 to 5 days, and particularly 4 days.

[0096] The culturing period in the medium containing FGF9 and heparin is, for example, 1 to 3 days, and particularly 1 day before single cell dissociation, and is, for example, 0.5 to 2 days, and particularly 2 days after single cell dissociation.

[0097] The concentration of the GSK3β inhibitor in step ii is, for example, 0.1 to 30 μM, preferably 1 to 10 μM, more preferably 3 to 7 μM, and particularly preferably 5 μM.

[0098] The concentration of FGF9 is, for example, 0.1 ng / ml to 1 μg / ml, preferably 1 to 500 ng / ml, more preferably 10 to 300 ng / ml, and particularly preferably about 200 ng / ml.

[0099] The concentration of heparin is, for example, 0.01 to 100 μg / ml, preferably 0.1 to 10 μg / ml, and particularly preferably about 1 μg / ml.

[0100] The culturing period in the medium containing the GSK3β inhibitor is, for example, 0.5 to 2 hours, and particularly 1 hour.

[0101] The culturing period in the medium containing FGF9 and heparin is, for example, 0.5 to 2 days, and particularly 1 day.

[0102] After step ii, the steps of culturing the renal organoids in the medium containing FGF9 and heparin and then culturing the renal organoids in the medium containing heparin can be carried out.

[0103] The culturing period in the medium containing FGF9 and heparin is, for example, 3 to 7 days, and particularly 4 days.

[0104] The culturing period in the medium containing heparin is, for example, 7 to 25 days, and particularly 14 days.

[0105] The concentrations of FGF9 and heparin can be the same as those used in step ii.

[0106] To confirm the generation of intermediate mesoderm and kidney organoids, for example, methods for measuring the expression of marker proteins or marker genes can be employed.

[0107] When OSR1 is expressed in the obtained cell aggregates, the cell aggregates can be determined as intermediate mesoderm.

[0108] When the obtained cell aggregates have the ability to produce EPO under hypoxic conditions and express markers of renal interstitial cells, glomeruli, and renal proximal tubules, the cell aggregates can be determined as kidney organoids. Examples of renal interstitial cell markers include FOXD1, PDGFRβ, and CD73. Examples of glomerular markers include WT1 and NPHS1. Examples of renal proximal tubule markers include LTL, CUBN, and E-cadherin.

[0109] In addition, the generation of intermediate mesoderm can also be confirmed by, for example, confirming the ability of cells to induce differentiation into glomeruli or renal proximal tubules.

[0110] [GSK3β inhibitor]

[0111] Examples of GSK3β inhibitors include CHIR98014 (N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine), CHIR99021 (6-{2-[4-(2,4-dichloro-phenyl)-5-(5-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino]-ethylamino}-nicotinonitrile), CP21R7 (CP21R7), LY2090314 (3-[9-fluoro-1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1h-pyrrole-2,5-dione), TDZD-8 (2-methyl-4-(phenylmethyl)-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), TWS-119 (3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol), camparol, 1-azacamparol, SB415286 ([3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H pyrrole-2,5-dione]) and AR-AO144-18 (1-[(methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea), CT20026, BIO ((2'Z,3'E)-6-bromoindirubin-3'-oxime), BIO-acetoxime, pyridino-carbazole-cyclopentadienyl ruthenium complex, OTDZT, α-4-dibromoacetophenone and lithium. Combinations of two or more of these can be used.

[0112] GSK3β inhibitors are not limited to these, and antisense oligonucleotides or siRNAs against the mRNA of GSK3β, antibodies that bind to GSK3β, dominant negative GSK3β mutants, etc. can be used as GSK3β inhibitors, and these are all commercially available or can be synthesized according to any known method.

[0113] [Culture medium]

[0114] There are no particular restrictions on the basal medium, and for example, STEMdiff APEL2 medium (STEMCELL Technologies, ST-05275), TeSR1 medium, and chemically defined medium (CDM) are suitable for use. In addition, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, modified MEM (IMEM) medium, modified MDM (IMDM) medium, Medium 199 medium, Eagle's MEM medium, αMEM medium, DMEM medium (high glucose, low glucose), DMEM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof can also be used.

[0115] There are no particular restrictions on the CDM medium, and for example, a medium prepared from Iscove's modified Dulbecco's medium (manufactured by GE Healthcare) can be used.

[0116] The basal medium can be supplemented with substances commonly used in cell culture, such as Ham's F-12 nutrient mixture, albumin (including human serum albumin), polyvinyl alcohol (PVA), deionized BSA, linoleic acid, linolenic acid, cholesterol, insulin, apo-transferrin, selenium, ethanolamine, monothioglycerol, protein-free hybridoma medium II (PFHMII), ascorbic acid, L-alanyl-L-glutamine, and / or antibiotics.

[0117] [Ratio between glomerular epithelial cell-like cells and renal proximal tubular epithelial cell-like cells]

[0118] When changing the addition period and concentration of the GSK3β inhibitor in step i, renal organoids with different ratios between glomerular epithelial cell-like cells and renal proximal tubular epithelial cell-like cells can be obtained.

[0119] Specifically, when inducing renal organoids rich in and having a high ratio of glomerular epithelial cell-like cells, the culture is carried out in a basal medium supplemented with 9.0 to 10.5 μM (preferably 9.5 to 10.5 μM) of the GSK3β inhibitor for 4 days, or in a basal medium supplemented with 7 to 9 μM (preferably 7.5 to 8.5 μM) of the GSK3β inhibitor for 5 days.

[0120] When inducing renal organoids rich in and having a high ratio of renal proximal tubule epithelial cell-like cells, the culture is carried out in a basal medium supplemented with 5.5 to 7.0 μM (preferably 5.5 to 6.5 μM) of a GSK3β inhibitor for 4 days, or in a basal medium supplemented with 6.5 to 8.5 μM (preferably 7.5 to 8.5 μM) of a GSK3β inhibitor for 3 days.

[0121] When the culture in step i is carried out in a basal medium supplemented with a GSK3β inhibitor at a concentration greater than 7.0 μM and less than 9.0 μM for 4 days, renal organoids containing an equal amount of glomerular epithelial cell-like cells and renal proximal tubule epithelial cell-like cells can be induced.

[0122] When using intermediate conditions between the addition period conditions and the GSK3β inhibitor concentration for inducing renal organoids rich in glomerular epithelial cell-like cells and the addition period conditions and the GSK3β inhibitor concentration for inducing renal organoids rich in renal proximal tubule epithelial cell-like cells, renal organoids having an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells are obtained.

[0123] During the remaining period after the addition of the GSK3β inhibitor is completed in step i, the culture is carried out in a medium containing FGF9 and heparin.

[0124] In renal organoids rich in glomerular epithelial cell-like cells, 30% to 80% of the cells contained in the renal organoids can be glomerular epithelial cell-like cells.

[0125] In renal organoids rich in renal proximal tubule epithelial cell-like cells, 5% to 30% of the cells contained in the renal organoids can be renal proximal tubule epithelial cell-like cells.

[0126] [Contact step]

[0127] The step of contacting the test substance with the renal organoids can be carried out by adding the test substance to the medium maintaining the renal organoids in a suitable container (such as a culture dish, flask or microplate). The step of contacting the test substance with the renal organoids can be carried out by, for example, introducing the basal medium containing the test substance into the gas phase and / or liquid phase of a transwell plate in which the renal organoids are prepared at the air-liquid interface.

[0128] [Measurement of cytotoxicity]

[0129] The cytotoxicity of the renal organoids that have been contacted with the test substance can be measured and evaluated according to any conventionally known method. Known methods are, for example, methods using a decrease in the amount of intracellular ATP, an increase in the amount of intracellular enzymes leaking into the extracellular space, or an increase in the amount of labeled proteins secreted into the medium as an index of cytotoxicity.

[0130] [Determination of Nephrotoxicity]

[0131] It can be determined that a test substance showing cytotoxicity to renal organoids may have nephrotoxicity.

[0132] More preferably, it can be determined that a test substance showing higher cytotoxicity compared to a control substance known to have no nephrotoxicity or low nephrotoxicity may have nephrotoxicity.

[0133] The determination criteria can be appropriately set according to the evaluation purpose and the type of substance to be evaluated. For example, when the index is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher than the control substance, and preferably 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more higher, it is determined that the substance has cytotoxicity.

[0134] In the method for evaluating the nephrotoxicity of a test substance according to the present disclosure, different from the in vitro evaluation method using a renal proximal tubule epithelial cell line (RPTEC), renal organoids containing both glomerular epithelial cell-like cells and renal proximal tubule epithelial cell-like cells are used, and thus, the in vivo toxicity of the test substance can be more appropriately reflected in the evaluation.

[0135] Furthermore, in the method for evaluating the nephrotoxicity of a test substance according to the present disclosure, when renal organoids having different ratios between glomerular epithelial cell-like cells and renal proximal tubule epithelial cell-like cells are used, it is possible to evaluate which one of the glomerulus and the renal proximal tubule the test substance shows higher toxicity to.

[0136] 2. Method for Screening Nephrotoxicity of Test Substances

[0137] The method for evaluating the nephrotoxicity of a test substance according to the present disclosure can be used in a method for screening test substances that may have nephrotoxicity or test substances that may have no nephrotoxicity or low nephrotoxicity.

[0138] The cytotoxicity of a variety of test substances is evaluated by the above-mentioned nephrotoxicity evaluation method, and a test substance showing cytotoxicity to renal organoids (more preferably, a test substance showing higher cytotoxicity compared to a control substance) is selected as a substance that may have nephrotoxicity.

[0139] Alternatively, the cytotoxicity of a variety of test substances is evaluated by the above-mentioned nephrotoxicity evaluation method, and a test substance showing no cytotoxicity to renal organoids or a test substance showing lower cytotoxicity compared to a control substance is selected as a substance that may have no nephrotoxicity or lower nephrotoxicity. This screening can be used to screen the nephrotoxicity of drug candidate compounds.

[0140] In addition, when using kidney organoids having different ratios between glomerular epithelial cell-like cells and renal proximal tubule epithelial cell-like cells, a screening system focused on the toxicity to one of the glomerulus and the renal proximal tubule can be obtained.

[0141] 3. Method for predicting the site where a test substance shows toxicity in the kidney

[0142] The method for evaluating the renal toxicity of a test substance according to the present disclosure can also be used in a method for predicting the site where a test substance shows toxicity in the kidney.

[0143] Perform step (A) of contacting the test substance with kidney organoids rich in glomerular epithelial cell-like cells and step (B) of contacting the test substance with kidney organoids rich in renal proximal tubule epithelial cell-like cells.

[0144] Then, in step (C), compare the cytotoxicity obtained in step (A) and the cytotoxicity obtained in step (B), and it can be predicted that when the cytotoxicity obtained in step (A) is higher, the site showing toxicity is the glomerulus, and when the cytotoxicity obtained in step (B) is higher, the site showing toxicity is the renal proximal tubule.

[0145] 4. Kit

[0146] The present disclosure also provides a kit including kidney organoids, which is used for the method for evaluating the renal toxicity of a test substance, the method for screening the renal toxicity of a test substance, and the method for predicting the site where a test substance shows toxicity in the kidney.

[0147] The kit according to the present disclosure may include kidney organoids rich in glomerular epithelial cell-like cells or kidney organoids rich in renal proximal tubule epithelial cell-like cells.

[0148] In addition to including kidney organoids, the kit according to the present disclosure may further contain a culture medium and reagents (such as GSK3β inhibitor, FGF, and heparin) for culturing kidney organoids, and reagents for measuring the cytotoxicity to kidney organoids.

[0149] The kit according to the present disclosure may include pluripotent stem cells and a culture medium and reagents (such as GSK3β inhibitor, FGF, and heparin) for inducing cell differentiation into kidney organoids to replace the kidney organoids.

[0150] Examples

[0151] Differentiation induction of induced pluripotent stem cell (iPSC)-derived kidney organoids (KiO) (Overview)

[0152] (1) Differentiation induction of iPSC into intermediate mesoderm spheroids

[0153] Step 1 (Day 1): Inoculation of iPSCs

[0154] Inoculate 1231A3 iPSCs at 5.76×10 5 cells / well into a 6-well plate coated with laminin (iMatrix-511 silk: Nippi Inc.). Incubate the resulting product overnight in an incubator at 37 °C and 5% CO2.

[0155] As the medium, AK03N (Ajinomoto Co., Inc.) supplemented with 10 μM Y27632 was used at a volume of 2 ml / well.

[0156] Step 2 (Days 0 to 7): Induction of intermediate mesoderm spheroids

[0157] Replace the medium with intermediate mesoderm differentiation medium. As the basal medium for the intermediate mesoderm induction medium (KiO differentiation induction basal medium), APEL2 medium (STEMCELL Technologies) supplemented with 5% PFHM (protein-free hybridoma medium: ThermoFisher) was used.

[0158] Step 2-1 (Days 0 to 3 / 4 / 5): In the first half of the period from Day 0 to Day 6, use the medium obtained by adding CHIR99021 (CHIR) to the KiO differentiation induction basal medium as the intermediate mesoderm differentiation medium.

[0159] Step 2-2 (Days 3 / 4 / 5 to Day 6): In the second half of the period from Day 0 to Day 6, use the medium obtained by adding 200 ng / ml FGF9 and 1 μg / ml heparin to the KiO differentiation induction basal medium as the intermediate mesoderm differentiation medium.

[0160] Replace the medium every two days.

[0161] Appropriately change the number of days in the first half period (using CHIR) and the second half period (not using CHIR) and the concentration of CHIR added in the first half period for the inspections described in the following examples.

[0162] Step 2-3 (Days 6 to 7): Formation of intermediate mesoderm spheroids using a 96-well plate

[0163] Dissociate the cells on Day 6 of differentiation induction into single cells with Accutase, and suspend the resulting product in the basal medium (KiO differentiation induction basal medium) supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin, and at 5×104 Cells (number per well: 1 cell / well, medium: 200 μl / well) were inoculated into a Prime Surface 96-well plate (Sumitomo Bakelite Co., Ltd.). The resulting plate was centrifuged at 300 g for 3 minutes to pellet the cells, and the cells were cultured overnight (for 1 day) in an incubator at 37 °C and 5% CO₂.

[0164] (2) Inducing KiO by air-liquid interface culture (ALIC)

[0165] Step 3 (day 7): Air-liquid interface culture using a Transwell plate

[0166] The intermediate mesoderm spheroids generated in the Prime Surface 96-well plate were washed by transferring them to a fresh plate of the same type (basal medium: 200 μl / well). After washing, the intermediate mesoderm spheroids were transferred to the insert of a Transwell plate. Basal medium supplemented with 5 μM CHIR was added to the corresponding wells of the Transwell plate to form the liquid phase. The insert was inserted into the well and allowed to stand for 1 hour, during which the intermediate mesoderm spheroids held in the insert were maintained at the air-liquid interface in an incubator at 37 °C and 5% CO₂. Subsequently, the medium in the liquid phase was replaced with basal medium supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin, and the resulting product was cultured overnight. As the Transwell plate, a 6-well Transwell plate or a 24-well Transwell plate was used.

[0167] Figure 1 The experimental protocols of steps 2-3 and 3 are shown. When the method of the present invention is adopted, uniform intermediate mesoderm spheroids can be generated, which vary little in terms of the type, number, and size of the cells constituting the spheroids, and the operation of keeping the intermediate mesoderm spheroids stationary at the air-liquid interface of the Transwell plate can be stably performed.

[0168] Step 4 (days 8 to 11): Culture supplemented with FGF9 and heparin

[0169] From day 8 to day 11, culture was carried out with basal medium supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin added to the liquid phase. The medium was changed every two days.

[0170] Step 5 (days 12 to 25): Culture supplemented with heparin

[0171] On day 12 and thereafter, culture was carried out with basal medium supplemented with 1 μg / ml heparin added to the liquid phase. The medium was changed every two days.

[0172] [Example 1: Examine the effect of the addition time period and concentration of CHIR on the ratio between glomerular epithelial cell-like cells and renal proximal tubular epithelial cells in KiO]

[0173] Change the addition time period and concentration of CHIR in Step 2 outlined above to examine how the ratio between glomerular epithelial cell-like cells and renal proximal tubular epithelial cell-like cells changes.

[0174] As conditions for the addition time period and concentration of CHIR, the following were examined:

[0175] Conditions for the addition time period of CHIR (see Figure 2 )

[0176] Condition (1): First half period (using CHIR): 3 days (Day 0 to Day 2) / Second half period (not using CHIR): 3 days (Day 3 to Day 5)

[0177] Condition (2): First half period: 4 days (Day 0 to Day 3) / Second half period: 2 days (Day 4 to Day 5) or

[0178] Condition (3): First half period: 5 days (Day 0 to Day 4) / Second half period: 1 day (Day 5)

[0179] Conditions for the addition concentration of CHIR:

[0180] 6 μM, 8 μM, and 10 μM.

[0181] Cultivation without CHIR was carried out in a medium obtained by supplementing a basal medium with 200 ng / ml FGF9 and 1 μg / ml heparin.

[0182] Step 1 (Day 1) and Steps 3 to 6 (Day 6 to Day 24) were carried out in the same manner as in Example 1. However, as the air-liquid interface cultivation in Steps 4 to 6 (Day 7 to Day 24), a 6-well transwell plate made of PET was used.

[0183] Figure 3 Fluorescence microscopy images of KiO induced under the same conditions where the addition concentration of CHIR was set to 8 μM and the addition time period of CHIR was changed among Conditions (1), (2), and (3) are shown. When Condition (1) (adding CHIR for 3 days) was adopted, KiO with renal proximal tubular epithelial cell-like cells expressing the renal proximal tubular marker LTL (red) at a high ratio was obtained (see Figure 3(A)). On the other hand, when condition (3) (addition of CHIR for 5 days) was adopted, KiO of glomerular epithelial cell-like cells with a high ratio of WT1 (green) expressing glomerular epithelial cell markers was obtained (see Figure 3 (C)). When condition (2) (addition of CHIR for 4 days) was adopted, KiO with an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells was obtained (see Figure 3 (B)).

[0184] Figure 4 Fluorescence microscopy images of KiO induced when the addition period of CHIR was set to condition (2) (addition of CHIR for 4 days) and the addition concentration of CHIR varied between 6 μM, 8 μM, and 10 μM are shown. When the addition concentration was 6 μM, KiO of renal proximal tubule epithelial cell-like cells with a high ratio of LTL (red) expressing renal proximal tubule markers was obtained (see Figure 4 (A)). On the other hand, when the addition concentration was 10 μM, KiO of glomerular epithelial cell-like cells with a high ratio of WT1 (green) expressing glomerular epithelial cell markers was obtained (see Figure 4 (C)). When the addition concentration was 8 μM, KiO with an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells was obtained (see Figure 4 (B)).

[0185] In KiO obtained at a CHIR addition concentration of 6 μM, a large number of cells in the form of renal proximal tubule-like were observed (see Figure 5 red dotted line).

[0186] Figure 6 Gene expression patterns of KiO obtained at CHIR addition concentrations of 6 μM, 8 μM, and 10 μM are shown. When the addition concentration was 6 μM, the expression of marker genes of renal proximal tubule epithelial cells (proximal tubule) and loop of Henle / distal renal tubule (loop of Henle / distal tubule) tended to be high. On the other hand, when the addition concentration was 10 μM, the expression of marker genes of glomerular epithelial cell-like cells (podocytes) tended to be high.

[0187] [Example 2: Air-liquid interface culture using a 24-well Transwell plate]

[0188] It was examined whether a 24-well transwell plate could be used in the air-liquid interface culture carried out in step 3 (day 7) and afterwards as outlined above.

[0189] Steps 1 and 2 (days 1 to 6) are carried out in the same manner as described in the overview.

[0190] The intermediate mesoderm spheroids generated in a Prime Surface 96-well plate are washed by transferring the intermediate mesoderm spheroids to a fresh plate of the same type (basic medium: 200 μl / well). After washing, the intermediate mesoderm spheroids are transferred to the insert of a 24-well transwell plate (Kurabo Industries Ltd.) made of PTFE (polytetrafluoroethylene) membrane material. The basic medium supplemented with 5 μM CHIR is added to the corresponding wells of the transwell plate to form the liquid phase (300 μl / well). The insert is inserted into the well and allowed to stand for 1 hour, with the intermediate mesoderm spheroids held in the insert remaining at the air-liquid interface in an incubator at 37 °C and 5% CO2. Subsequently, the medium of the liquid phase is replaced with the basic medium supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin, and the resulting product is cultured overnight.

[0191] Steps 4 and 5 (days 8 to 24) are carried out in the same manner as described in the overview.

[0192] When using a 24-well transwell plate, the operation of placing the intermediate mesoderm spheroids in the insert can be carried out more stably compared to when using a 6-well transwell plate. In addition, KiO can be stably prepared without the medium penetrating into the gas phase. It was confirmed that the KiO prepared in the 24-well transwell plate has substantially the same gene expression pattern as the KiO prepared by the conventional method using a 6-well transwell plate (see Example 1 and Non-Patent Document 2) (no data).

[0193] [Example 3: Toxicity evaluation using KiO]

[0194] (1) Addition of test substance

[0195] According to the method described in Example 2, the KiO (day 25) generated at the air-liquid interface of a 24-well transwell plate is exposed to the test substance. Specifically, the basic medium supplemented with the solvent containing the test substance (300 μl / well) is introduced into the gas phase of the transwell plate to replace the medium of the liquid phase with the basic medium supplemented with the test substance (500 μl / well). The resulting product is cultured in an incubator at 37 °C and 5% CO2 for 3 days to expose the KiO to the test substance.

[0196] (2) Cytotoxicity measurement 1: Measurement of intracellular ATP amount

[0197] The cytotoxicity of a test substance is measured based on the amount of ATP in cells.

[0198] Specifically, KiO that had been exposed to the test substance for 3 days was transferred from a transwell to a 1.5-ml tube, and the resulting material was centrifuged at 300 g for 5 minutes, and then the supernatant was removed. KiO was lysed in 100 μl of CellTiter-Glo 3D reagent (Promega), and further sonicated to completely lyse the cells. The cell lysate thus obtained was transferred to a 96-well plate and allowed to stand at 25 °C in the dark for 25 minutes. The amount of luminescence dependent on the amount of ATP was measured using a microplate reader.

[0199] As a control substance, the solvent alone was used.

[0200] The cytotoxicity was calculated as "(measured value of the test substance / measured value of the control substance) × 100%".

[0201] The results obtained by using cyclosporin A (concentration: 1 × 10 -7 M to 1 × 10 -3 M) as the test substance are shown in Figure 7 . It is known that cyclosporin A shows strong toxicity in glomeruli. In KiO produced by the method described in Example 3, cytotoxicity dependent on the concentration of cyclosporin A was detectable. On the other hand, when a similar test was performed using a renal proximal tubule epithelial cell line (RPTEC) instead of KiO, cytotoxicity dependent on the concentration of cyclosporin A was not detectable.

[0202] In addition, according to the method described in Example 1, KiO having a high ratio of renal proximal tubule epithelial cell-like cells, KiO having a high ratio of glomerular epithelial cell-like cells, and KiO having an intermediate ratio between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells were produced and tested similarly. The results are shown in Figure 8 . In KiO having a high ratio of renal proximal tubule epithelial cell-like cells (rich in tubules), higher cytotoxicity was detected, and in KiO having a high ratio of glomerular epithelial cell-like cells (rich in glomeruli), lower cytotoxicity was detectable. When KiO with different ratios between renal proximal tubule epithelial cell-like cells and glomerular epithelial cell-like cells produced by the method described in Example 1 was used, the cytotoxicity characteristics of cyclosporin A (strong cytotoxicity in glomeruli) could thus be detected.

[0203] (3) Cytotoxicity measurement 2: Measurement of LDH leakage amount

[0204] The cytotoxicity of a test substance is measured based on the amount of LDH leaked from cells.

[0205] Specifically, after exposing the KiO produced according to the method described in Example 1 to the test substance for 3 days, 100 μl of the culture medium (liquid phase) was collected into a 96-well plate. The obtained product was mixed with 100 μl of the LDH measurement buffer (Cytotoxicity LDH Assay Kit - WST: DOJINDO LABORATORIES), and the resulting mixture was allowed to stand in an incubator at 37 °C and 5% CO2 for 30 minutes. 50 μl of the termination solution was added thereto to terminate the reaction, and the absorbance at 490 nm was measured using a microplate reader.

[0206] As a control substance, the solvent was used alone.

[0207] The cytotoxicity was calculated as "(measured value of the test substance / measured value of the control substance) × 100%".

[0208] The results are shown in Figure 9 . In all KiO, cytotoxicity dependent on the concentration of cyclosporin A was observed. In addition, when using KiO with a high ratio of glomerular epithelial cell-like cells (rich in glomeruli), toxicity was easily observed.

[0209] (4) Cytotoxicity measurement 3: Measurement of secreted labeled protein

[0210] The cytotoxicity of the test substance was measured based on the amount of the nephrotoxic marker KIM-1 (kidney injury marker 1) secreted into the culture medium.

[0211] Quantitative determination of KIM-1 was performed using a KIM-1 ELISA kit (R&D). The KiO produced according to the method described in Example 1 was exposed to the test substance for 3 days, then 50 μl of the culture medium (liquid phase) was collected and transferred into a 96-well plate in which an anti-KIM-1 capture antibody was immobilized. The obtained product was allowed to stand in the dark at 25 °C for 2 hours. The obtained wells were washed four times with a cleaning solution containing a surfactant, and 200 μl of a buffer containing an anti-KIM-1 detection antibody was added thereto. The obtained product was allowed to stand in the dark at 25 °C for 2 hours. The obtained wells were washed four times with a cleaning solution containing a surfactant, and 200 μl of a substrate solution was added thereto. The obtained product was allowed to stand in the dark at 25 °C for 30 minutes. The reaction was terminated by adding 50 μl of the termination solution, and the absorbance at 450 nm was measured using a microplate reader.

[0212] As a control substance, the solvent was used alone.

[0213] The cytotoxicity was calculated as "(measured value of the test substance / measured value of the control substance) × 100%".

[0214] The results are shown inFigure 10 Similarly, when the amount of secreted KIM-1 is used as an index, the cytotoxicity of cyclosporin A can be detected.

[0215] [Example 4: Improved method for inducing differentiation of iPSC-derived KiO]

[0216] (1) Inducing differentiation of iPSC into intermediate mesoderm spheroids

[0217] Step 1 (Day 1): Seeding of iPSC

[0218] The seeding is carried out by the same method as described in the above overview.

[0219] Step 2 (Day 0 to Day 5): Induction of intermediate mesoderm spheroids

[0220] In the method described in the above overview, the period of Step 2-1 (the first half period) is set as Day 0 to Day 4, and the period of Step 2-2 (the second half period) is set as Day 4 to Day 5.

[0221] Replace the medium with intermediate mesoderm differentiation medium. As the basal medium for intermediate mesoderm induction medium (KiO differentiation induction basal medium), use the medium obtained by adding 5% PFHM (protein-free hybridoma medium: ThermoFisher) to APEL2 medium (STEMCELL Technologies).

[0222] Step 2-1 (Day 0 to Day 4): In the first half of the period from Day 0 to Day 5, use the medium obtained by adding CHIR99021 (CHIR) to the KiO differentiation induction basal medium as the intermediate mesoderm differentiation medium.

[0223] Step 2-2 (Day 4 to Day 5): In the second half of the period from Day 0 to Day 5, use the medium obtained by adding 200 ng / ml FGF9 and 1 μg / ml heparin to the KiO differentiation induction basal medium as the intermediate mesoderm differentiation medium.

[0224] Replace the medium every two days.

[0225] Step 2-3 (Day 5 to Day 7): Forming intermediate mesoderm spheroids using a 96-well plate

[0226] In this improved method, the period from Day 6 to Day 7 in Step 2-3 of the method described in the above overview is changed to the period from Day 5 to Day 7.

[0227] On the 5th day of differentiation induction, the cells were dissociated into single cells with Accutase, and the resulting product was suspended in a basal medium (KiO differentiation induction basal medium) supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin, and seeded at 5×10 4 cells / well (medium: 200 μl / well) in a Prime Surface 96-well plate (Sumitomo Bakelite Co., Ltd.). The resulting plate was centrifuged at 300 g for 3 minutes to pellet the cells, and the cells were cultured in an incubator at 37 °C and 5% CO2 for 2 days.

[0228] Since the culture period was increased from 1 day to 2 days, cell-centered spheroid solids (with a clear core) could be formed, and KiO could be induced more stably in subsequent step 3.

[0229] (2) Inducing KiO by air-liquid interface culture (ALIC)

[0230] Step 3 (Day 7): Air-liquid interface culture using a Transwell plate

[0231] The culture was performed in the same manner as described in the above overview, except that the membrane of the insert of the Transwell plate was pre-hydrophilized.

[0232] The intermediate mesoderm spheroids generated in the Prime Surface 96-well plate were washed by transferring them to a fresh plate of the same type (basal medium: 200 μl / well). After washing, the intermediate mesoderm spheroids were transferred to the insert of the Transwell plate. Two days before use, the insert of the Transwell plate was immersed in water (upper layer: 500 μl, lower layer: 1 ml) to hydrophilize the membrane. Inserts with poor hydrophilicity were not used. The basal medium supplemented with 5 μM CHIR was added to the corresponding wells of the Transwell plate to form a liquid phase. The insert was inserted into the well and allowed to stand for 1 hour, with the intermediate mesoderm spheroids retained at the air-liquid interface in the incubator at 37 °C and 5% CO2. Subsequently, the medium of the liquid phase was replaced with the basal medium supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin, and the resulting product was cultured overnight.

[0233] Since the membrane was hydrophilized, the extension of the spheroids on the membrane was accelerated, and thus, flatter spheroids with a smaller height could be formed in the insert. In this way, necrosis of the cells in the higher positions (positions far from the membrane surface) present in the spheroids due to insufficient nutrient supply from the air-liquid interface could be inhibited.

[0234] Step 4 (Day 8 to Day 11): Culture supplemented with FGF9 and heparin

[0235] Step 5 (Day 12 to Day 25): Culture supplemented with heparin

[0236] These steps are carried out in the same manner as the method described in the above overview, except that an operation for washing away necrotic cells is performed.

[0237] From Day 8 to Day 11, culture is carried out using a basal medium supplemented with 200 ng / ml FGF9 and 1 μg / ml heparin added to the liquid phase. The culture medium is changed every two days. On Day 12 and thereafter, culture is carried out using a basal medium supplemented with 1 μg / ml heparin added to the liquid phase. The culture medium is changed every two days.

[0238] Once a week, water is dripped onto the KiO from above to wash away necrotic cells. In this way, the viability of the cells included in the KiO is improved, and the visibility of the KiO is also improved.

Claims

1. A method for evaluating the nephrotoxicity of a test substance, the method comprising: The step of contacting the test substance with renal organoids, wherein higher cytotoxicity of the test substance compared to a control substance indicates that the test substance may have nephrotoxicity.

2. A method for screening a test substance that may have nephrotoxicity, the method comprising the following steps: (1) Contacting the test substance with renal organoids; and (2) Selecting a test substance that shows higher cytotoxicity compared to a control substance.

3. The method according to claim 1 or 2, wherein the renal organoids are derived from human induced pluripotent stem cells.

4. The method according to claim 1 or 2, wherein the renal organoids are rich in glomerular epithelial cell-like cells.

5. The method according to claim 4, wherein 30% to 80% of the cells contained in the renal organoids are glomerular epithelial cell-like cells.

6. The method according to claim 4, the method further comprising: Step (a) of culturing pluripotent stem cells in the presence of a 9.0 to 10.5 μM GSK3β inhibitor to obtain the renal organoids rich in glomerular epithelial cell-like cells.

7. The method according to claim 1 or 2, wherein the renal organoids are rich in renal proximal tubule epithelial cell-like cells.

8. The method according to claim 7, wherein 5% to 30% of the cells contained in the renal organoids are renal proximal tubule epithelial cell-like cells.

9. The method according to claim 8, the method further comprising: Step (b) of culturing pluripotent stem cells in the presence of a 5.5 to 8.5 μM GSK3β inhibitor to obtain the renal organoids rich in renal proximal tubule epithelial cell-like cells.

10. A method for predicting the site where a test substance shows toxicity in the kidney, the method comprising the following steps: (A) Contacting the test substance with renal organoids rich in glomerular epithelial cell-like cells; (B) Contacting the test substance with renal organoids rich in renal proximal tubule epithelial cell-like cells; and (C) Comparing the cytotoxicity obtained in step (A) with the cytotoxicity obtained in step (B) to predict that the site showing toxicity is the glomerulus when the cytotoxicity obtained in step (A) is higher, and predicting that the site showing toxicity is the renal proximal tubule when the cytotoxicity obtained in step (B) is higher.

11. A method for screening the nephrotoxicity of a drug candidate compound, the method comprising the following steps: (1) Contacting the test substance with renal organoids rich in glomerular epithelial cell-like cells; and (2) Selecting a test substance that shows no cytotoxicity or lower cytotoxicity compared to a control substance as the drug candidate compound.

12. A kit for evaluating the nephrotoxicity of a test substance, the kit comprising renal organoids.

13. A method for generating renal organoids rich in glomerular epithelial cell-like cells, the method comprising: Step (a) of culturing pluripotent stem cells in the presence of a 9.0 to 10.5 μM GSK3β inhibitor.

14. A method for generating kidney organoids rich in kidney proximal tubule epithelial cell-like cells, the method comprising: Step (b) of culturing pluripotent stem cells in the presence of a GSK3β inhibitor at 5.5 to 8.5 μM.

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